Phosphorylation of visual pigments is essential for normal vision. Deficiencies in this biochemical reaction can cause night blindness and photoreceptor apoptosis. Phosphorylation uncouples rhodopsin and most G-protein coupled receptors from downstream signaling pathways. The sites of phosphorylation on most G-protein coupled receptors are not known. During the previous funding period we developed a mass- spectrometry based method that enables us to determine the sites and kinetics of phosphorylation and dephosphorylation reactions that occur on visual pigments in intact retinas. We have used the method to establish the sites and kinetics of phosphorylation on mouse and zebrafish visual pigments under a variety of physiological conditions. We propose to use the method to pursue four new specific aims. 1. Evaluate rhodopsin phosphorylation during long-term illumination and identify relationships between visual pigment phosphorylation, visual cycle and visual sensitivity. 2. Evaluate factors that influence phosphorylation of cone visual pigments. 3. Identify structural characteristics that determine site selection and kinetics of phosphorylation by rhodopsin kinase. 4. Evaluate the influence of recoverin and Calcium on visual pigment phosphorylation. These studies will provide the most precise data yet available about the kinetics and site preferences for phosphorylation of G-protein coupled receptors.